Enhanced Diagnostic Tools
Need an efficient way to prepare proteins with variation at specific sites? Bio-Synthesis
offers trimer oligonucleotide synthesis services using 2'-deoxynucleoside
trinucleotides. This protein engineering technique uses oligonucleotides of mixed
sequences to generate libraries of proteins that are often used to screen potential
improvements in specific biological function.
Trimer oligonucleotide synthesis has proven to be extremely valuable because they
allow codon-based mutagenesis, which circumvents the common problems of codon-bias,
frame-shift mutations, and the introduction of nonsense or stop codons.1
Trimer codon oligonucleotide synthesis is accomplished by using trinucleotide chemical
synthesis of 3 nucleotides in each synthesis cycle. This trimer oligonucleotide
synthesis introduces a mixture of all 20 amino acid codons (or subset) at any location
within the sequenced to be mutated. This leads to the production of clonal libraries
of exceptional diversity with order-of-magnitude increases in amino acid sequence
variance while either maintaining a uniform amino acid distribution 2
or one that is biased toward a desired set of amino acids.3
Inquire for Custom Trimer Oligonucleotide Synthesis.
The ability to efficiently and economically generate libraries of defined pieces
of DNA is the key strategy used for protein engineering research. These pools of
DNA molecules of related but non-identical sequences random gene library are often
used to express in an appropriate vector to generate the protein set encoded by
the library, and then screening the expressed proteins to identify protein-binding
sites in select experiment, enzyme selectivity, or protein stability.
While a variety of site-directed mutagenesis methods have been used to introduce
randomization at specific base positions of a gene, oligonucleotide-directed mutagenesis
is probably the most popular approach for the preparation of proteins with variations
at specific sites. One of the popular choices is to make pools of degenerate oligonucleotides,
which can be incorporated into the genes as cassettes or by PCR by using the degenerate
oligo as a primer. Degenerate oligonucleotides are synthesized as a mixture of A/C/G/T
phosphoramidites (N) at the site of the codons to be mutated. Since coupling efficiency
of DNA base react differently during chemical synthesis, this method often introduces
significant amounts of undesirable codon bias, frame-shift mutations, and stop codons
into the library, as result, mixed-base degenerate oligonucleotide preparation often
leaves very little control to achieve ratios of codons for specific amino acids,
therefore, does not always result in expected usage.
The problem of unwanted codon bias can be eliminated by synthesizing the random
portion of the oligos codon-by-codon using trimer codon synthesis method. This codon-based
oligonucletoide-directed mutagenesis system conveys the ability to effectively control
codon bias in the system, as well as unwanted stop codons and frame-shift mutations.
The researcher can choose to completely randomize a particular amino acid position
in the expressed protein, or flexibly tune the relative amounts of different amino
acids at that position, as needs require3. The trimer codon oligonucleotide
synthesis method has been used to develop phage display libraries with greater diversity
(as much as 10x greater) than by traditional methods, with a high degree of amino
acid uniformity4 . Trimer codon oligonucleotides were also used to maximize
the diversity of a partially randomized library for use in developing streptavidin
variants with altered specificities for desthiobiotin, a biotin analog, by directed
evolution5.The combination of standard and trimer codon phosphoramidite mixes is
a powerful research tool for anyone doing protein engineering research.
Bio-Synthesis offers sense trimers and antisense trimers at two different pre-mixes
or individual trimer codon for custom preparation.
While Trimer oligonucleotide synthesis has proven to be a useful tool for codon-based
mutagenesis. However, difficulties arise when long oligonucleotides are required
to introduce mutations in multiple distal regions of a gene simultaneously. Long
oligonucleotides often lead to lower sequence fidelity due to deletion mutants,
depurination events or mutations cause by deamination of cytidine. This can be accommodated
by using antisense trimer codon amidite. These trimers are the reverse complement
of the cannonical 'sense' codons. When these antisense codons are put into the noncoding
strand of a template DNA and amplified by PCR, they will code for the sense codon
in the opposite strand of DNA. This allows the powerful technique of PCR Assembly8
to generate not only kilobase-sized genes from short 50mer oligonucleotides, but
to simultaneously mutate multiple distal regions of that gene as shown in image
below for simultaneous mutation of multiple distal regions of gene.
The sense and their corresponding antisense codons are listed in Table 1. Conveniently,
many of our existing sense trimers can act as antisense codons. For example, AAC,
which codes for asparagine, has the anticodon GTT, which is the sense codon for
valine. However, some of the existing trimers, while they can act as an antisense
codon, are not good choices for use. For example, TGG, which codes for tryptophan,
could be used as an antisense codon for proline because CCA is one of proline's
synonymous codons. However, CCA has a relatively low Codon Adaptation Index (CAI)
value9 in E. coli, which could limit protein expression in that commonly
used organism. For this reason, the anticodon CGG was chosen for optimal expression
in E. coli, as were the other new antisense codons see, table below.